Kinetics and energetics of subunit dissociation/unfolding of TIM: the importance of oligomerization for conformational persistence and chemical stability of proteins.
Kinetics of unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM) were measured as a function of guanidine hydrochloride (GdnHCl) concentration. From the rate constants of these processes, the activation free-energy barriers (delta G++) were calculated using the Arrhenius equation. Assuming a linear dependence of delta G++ on the concentration of GdnHCl, activation energies in the absence of GdnHCl were estimated. The Gibbs free-energy change of dissociation/unfolding (delta G) was determined from GdnHCl unfolding curves in equilibrium. Using these data and the literature value for the bimolecular association rate constant of folded TIM monomers [Zabori, S., Rudolph, R., and Jaenicke, R. (1980) Z. Naturforsch. 35C, 999-1004], a model was developed that fully describes both kinetics and energetics of subunit dissociation/unfolding of TIM. Unfolded TIM monomers are susceptible to proteolytic digestion and thiol oxidation, while native TIM is resistant to both. The present model explains how the dimeric nature of TIM decreases the frequency of subunit unfolding by several orders of magnitude, thus increasing the chemical stability of the protein. Furthermore, the model also explains the recently demonstrated persistence (on a time scale of hours to days) of conformational heterogeneity of native TIM dimers [Rietveld, A. W. M., and Ferreira, S. T. (1996) Biochemistry 35, 7743-7751]. Again, it appears that the dimeric nature of TIM is essential for this behavior.
Rietveld
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Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil. rietveld@bioqmed.ufrj.br
Ferreira
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ST
D000818:Animals; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004734:Energy Metabolism / Q000187:drug effects; D019791:Guanidine / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism*
D011108:Polymers; D014305:Triose-Phosphate Isomerase; D019791:Guanidine